Leishmania parasites can persist for decades in infected humans, but the mechanisms responsible for their persistence and disease pathogenesis are not entirely clear. During the previous funding period, we found that L. amazonensis, especially its amastigote form, has evolved sophisticated strategies to evade recognition by dendritic cells (DC) and CD4+ T cells and to prevent activation of and destruction by macrophages. On the other hand, a low, but sustained, level of activation of pathogenic CD4+ T cells is required for disease progression. We hypothesized that L. amazonensis prevents the host from generating efficient protective immunity via two key mechanisms: subverting host autophagy machinery and apoptotic mimicry by exposing phosphatidylserine (PS) on parasite surface. These parasite strategies not only ensure their intracellular replication but also impede immune recognition through repressing antigen-presentation via the MHC class II pathway. This novel hypothesis will be tested in two complementary aims. Specific Aim 1 will examine how Leishmania parasites hijack and subvert host autophagic machinery and whether they hinder lysosomal fusion for impeding DC function. Our research strategies include the infection of autophagy-deficient mice and cells, the use of autophagy-inducing or -suppressing drugs, and the monitoring of autophagy dynamics by using tractable markers. Specific Aim 2 will dissect the mechanisms by which Leishmania evades host recognition and down-modulates immune responses via an apoptotic mimicry strategy. Our new data imply an involvement of macrophage-intrinsic and T cell-dependent mechanisms for parasite PS externalization. To distinguish their contributions, we will use mice deficient in the oxidative stress pathways (NADPH oxidase, glutathione peroxidase, and superoxide dismutase). We will monitor the level of PS exposure on parasites and DC antigen-presentation function during the course of in vitro and in vivo infection in the presence/absence of T cell cytokines, PS-neutralizing antibody, or newly developed anti-Leishmania drugs. For mechanistic investigations of parasite-related factors in both Specific Aims, we have parasite stocks derived from patients with localized or diffuse cutaneous leishmaniasis, as well as genetically modified parasites with reduced virulence or tractable markers. We seek continued support to carry this highly significant research forward. Completion of this study will provide new insight into the strategies utilized by this and other vesicle-dwelling pathogens for growth within the hostile environment of phagocytes, and aid in designing comprehensive approaches for the control of Leishmania and other intracellular infections for which efficient vaccines are not yet available.